WO2020256372A1 - Vecteur recombinant pour produire un antigène pour le diagnostic de la peste porcine africaine et son utilisation - Google Patents

Vecteur recombinant pour produire un antigène pour le diagnostic de la peste porcine africaine et son utilisation Download PDF

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WO2020256372A1
WO2020256372A1 PCT/KR2020/007762 KR2020007762W WO2020256372A1 WO 2020256372 A1 WO2020256372 A1 WO 2020256372A1 KR 2020007762 W KR2020007762 W KR 2020007762W WO 2020256372 A1 WO2020256372 A1 WO 2020256372A1
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swine fever
african swine
recombinant vector
polynucleotide encoding
protein
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PCT/KR2020/007762
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English (en)
Korean (ko)
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손은주
이상민
강향주
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주식회사 바이오앱
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Priority claimed from KR1020200072204A external-priority patent/KR102444024B1/ko
Application filed by 주식회사 바이오앱 filed Critical 주식회사 바이오앱
Priority to JP2020540527A priority Critical patent/JP7164894B2/ja
Priority to EP20739840.5A priority patent/EP3835425A4/fr
Priority to US16/964,629 priority patent/US20230287440A1/en
Priority to CN202080001357.5A priority patent/CN112424366B/zh
Publication of WO2020256372A1 publication Critical patent/WO2020256372A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention provides a recombinant vector for producing an antigen P32 protein for diagnosis of African swine fever (ASF), a transformant transformed with the recombinant vector, and an African containing P32 protein of the ASF virus isolated from the transformant It relates to a composition and kit for diagnosing swine fever.
  • ASF African swine fever
  • African swine fever is a swine infectious disease caused by infection with the African swine fever virus ( ASFV ) belonging to the Asfarviridae family.
  • ASFV African swine fever virus
  • African swine fever is a high-risk infectious disease that has a mortality rate of 100% when infected with pigs, and a vaccine against this disease has not been developed, so it is very important to diagnose it as an infectious disease with a high risk that must be disposed of immediately.
  • the present inventors have made diligent efforts to develop a rapid African swine fever antigen diagnosis method to maintain a clean African swine fever country and prepare for the influx of diseases.
  • the P32 recombinant protein among the African swine fever virus-specific proteins can be expressed with high efficiency in plants.
  • African swine fever virus is a large double-stranded DNA virus that replicates the cytoplasm of infected cells.
  • the virus continuously infects the genus Ornithodoros, a natural host pig, warthog, wild boar, and Ornithodoros, which can act as a vector without signs of disease, while causing a high mortality hemorrhagic fever in pigs.
  • This virus causes fatal hemorrhagic fever in pigs, so early diagnosis is very important.
  • the present invention was derived to solve the problems of the prior art and the necessity to quickly diagnose an individual suspected of being infected with African swine fever as described above, and not only enables efficient production using plants, but also diagnostic sensitivity. And a recombinant African swine fever virus antigen having high specificity, and a composition or kit for diagnosing African swine fever comprising the same.
  • an object of the present invention is to provide a recombinant vector containing a gene encoding the African swine fever virus antigen, and a transformant transformed by the recombinant vector.
  • the present invention comprises a polynucleotide encoding the African swine fever virus P32 protein consisting of the amino acid sequence of SEQ ID NO: 2, characterized in that it is expressed in plants. It provides a recombinant vector for antigen production for diagnosis of African swine fever.
  • the recombinant vector may further include a polynucleotide encoding a biP (chaperone binding protein) of SEQ ID NO: 3.
  • the polynucleotide encoding BiP may be located in the 5'end direction of the polynucleotide encoding the P32 protein, but is not limited thereto.
  • the recombinant vector may further include a polynucleotide encoding polyhistidine of SEQ ID NO: 4.
  • the polynucleotide encoding the polyhistidine may be located in the 3'end direction of the polynucleotide encoding the P32 protein, but is not limited thereto.
  • the recombinant vector may further include a polynucleotide encoding the HDEL of SEQ ID NO: 6.
  • the polynucleotide encoding the HDEL may be located in the 3'end direction of the polynucleotide encoding the P32 protein, but is not limited thereto.
  • the recombinant vector comprises a polynucleotide encoding BiP of SEQ ID NO: 3; A polynucleotide encoding the polyhistidine of SEQ ID NO: 4; And it may further include a polynucleotide encoding the HDEL of SEQ ID NO: 6, but is not limited thereto.
  • the recombinant vector may be a polynucleotide encoding BiP, a polynucleotide encoding a P32 protein, a polynucleotide encoding a polyhistidine, and a polynucleotide encoding an HDEL may be sequentially linked, It is not limited thereto.
  • the recombinant vector may include the nucleotide sequence of SEQ ID NO: 8, but is not limited thereto.
  • the present invention provides a transformant transformed with the recombinant vector.
  • the transformant may be a plant.
  • the present invention provides an African swine fever virus P32 recombinant protein produced using the recombinant vector.
  • the present invention provides a use of the African swine fever virus P32 recombinant protein produced using the recombinant vector for diagnosing African swine fever.
  • the present invention provides an African swine fever virus P32 recombinant protein produced using the recombinant vector for use in producing an agent used for diagnosis of African swine fever.
  • the protein may be water-soluble.
  • the present invention provides a composition for diagnosing African swine fever, comprising the recombinant protein of the African swine fever virus P32 as an active ingredient.
  • the present invention provides a kit for diagnosing African swine fever, comprising the recombinant protein of the African swine fever virus P32 as an active ingredient.
  • the present invention uses the recombinant protein of African swine fever virus P32 produced using the recombinant vector as an antigen, and provides an antibody against the African swine fever virus through an antigen-antibody reaction in a biological sample derived from animals other than humans. It provides a method for diagnosing African swine fever or determining African swine fever comprising the step of detecting.
  • the present invention comprises the steps of (a) transforming the recombinant vector according to the present invention into a plant; And (b) separating and purifying the recombinant antigen from the plant. It provides a method for producing a recombinant antigen for diagnosis of African swine fever.
  • the present invention comprises the steps of (a) transforming the recombinant vector according to the present invention into a plant; (b) separating and purifying the recombinant antigen from the plant; And (c) preparing a diagnostic composition or a diagnostic kit using the isolated/purified recombinant antigen, providing a method for preparing a composition or kit for diagnosing African swine fever.
  • Proteins for use in diagnosing and preventing viral diseases including African swine fever, especially antigens cannot use bacteria due to problems such as folding and glycosylation of proteins, and mainly using animal cells. Is being produced.
  • the vaccine production method using animal cells is not easy to manufacture because it takes a large cost to expand the facility for mass production, and in most cases, the price of the antigen is expensive.
  • antigens prepared using animal cells are not easy to store, and have a disadvantage in that they are highly likely to be contaminated with viruses that can infect animals.
  • the present invention compensates for this disadvantage by using plants. In other words, unlike animal cells, plant cells are unlikely to be contaminated with viruses that can infect animals, and can be mass-produced at any time as long as the cultivation area is secured, as well as long-term storage through the plant. This is possible.
  • the recombinant African swine fever virus antigen of the present invention is not only effectively expressed in plants, but also has high water solubility, so it is easy to separate and purify, and also, high sensitivity and specificity to the African swine fever virus. Since it shows specificity, it is expected to be widely used in various fields.
  • composition or kit capable of diagnosing African swine fever can be produced using the recombinant African swine fever virus antigen protein according to the present invention, and thus, an individual infected with African swine fever can be diagnosed early, thereby preventing the spread of the disease. It can be usefully used for
  • NB new chaperone binding protein
  • 6xHis polyhistidine tag
  • HDEL ER retention signal
  • FIG. 2 is a diagram showing the results confirmed by Western blotting after expressing the P32 antigen in a plant, separated and purified (T, Total extract; S, Supernatant fraction; P, Pellet fraction).
  • FIG. 3 is a view showing the result of confirming the purity after expressing the African swine fever virus P32 antigen in a plant according to an embodiment of the present invention.
  • Figure 4 is a result of preparing an antibody serum diagnostic kit for African swine fever comprising the composition of the present invention, and testing the reactivity and sensitivity of the antibody serum diagnostic kit using serum provided by the standard laboratory for African swine fever in Spain. It is a view showing.
  • the present invention can provide a recombinant vector for antigen production for diagnosis of African swine fever, which comprises a polynucleotide encoding the P32 protein of the African swine fever virus and is expressed in a plant.
  • the African swine fever virus P32 protein is known to be involved in internalization of the virus in the early stages of the infectious cycle.
  • the P32 protein may be encoded by the nucleotide sequence of SEQ ID NO: 1 or may be composed of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto. More specifically, the nucleotide sequence encoding the African swine fever virus P32 protein of the present invention may consist of a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto, and variants of the nucleotide sequence are included within the scope of the present invention. .
  • the nucleic acid molecule of the nucleotide sequence represented by SEQ ID NO: 1 of the present invention is a functional equivalent of the nucleic acid molecule constituting it, for example, some nucleotide sequences of the nucleic acid molecule are deleted, substituted, or inserted. It is a concept that includes variants that have been modified by, but are capable of functionally equivalent to nucleic acid molecules.
  • the gene is 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more sequence homology with the base sequence of the nucleotide sequence represented by SEQ ID NO: 1 It may include a nucleotide sequence having.
  • sequence homology includes polynucleotides having The “% of sequence homology” for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).
  • polynucleotide refers to an oligomer or polymer comprising two or more linked nucleotides or nucleotide derivatives linked to each other by a phosphate ester bond, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Show. Polynucleotides may also include, for example, nucleotide analogs, or "backbone” bonds other than phosphate ester bonds, such as phosphate hemp bonds, phosphoramidate bonds, phosphorothioate bonds, thioester bonds or peptide bonds (peptide DNA and RNA derivatives, including nucleic acids).
  • Polynucleotides include single-stranded and/or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs of either RNA or DNA.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the recombinant vector is a polynucleotide encoding BiP (Chaperone binding protein), a polynucleotide encoding 6 consecutive histidines (polyhistidine), or HDEL (His-Asp-Glu-Leu). ) It may further include a polynucleotide encoding a peptide.
  • the polynucleotide encoding BiP may be located in the 5'end direction of the polynucleotide encoding the P32 protein, and the polynucleotide encoding the polyhistidine and the polynucleotide encoding the HDEL peptide May be located in the 3'end direction of the polynucleotide encoding the P32 protein.
  • the term "recombinant vector” refers to a vector capable of expressing a peptide or protein encoded by a heterogeneous nucleic acid inserted into the vector, and preferably the target antigen (in the present invention, Africa It refers to a vector prepared to express porcine fever antigen P32).
  • the “vector” refers to an arbitrary medium for the introduction and/or transfer of a base into a host cell in vitro, in vivo or in vivo, and a replication unit capable of bringing about replication of the bound fragment by binding other DNA fragments ( replicon), and the term “replication unit” refers to any genetic unit (eg, plasmid, phage, cosmid, etc.) that functions as a self-unit of DNA replication in vivo, that is, can replicate by self-regulation. Chromosomes, viruses, etc.).
  • the recombinant vector of the present invention is preferably a promoter, which is a transcription initiation factor to which RNA polymerase binds, an arbitrary operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and termination of transcription and translation. It may include a sequence to control, a terminator, etc., more preferably a BiP gene, a hist-tag (His-tag, an amino acid motif consisting of at least 6 histidine residues), an endoplasmic reticulum signal peptide (endoplasmic reticulum signal peptide, endoplasmic reticulum targeting).
  • the same meaning as the sequence) may further include a gene, a cloning site, and the like, and more preferably may further include a selection marker gene such as an antibiotic resistance gene for selecting a transformant. .
  • the “gene (indicated by NB in FIG. 1)” is preferably a gene containing the nucleotide sequence of SEQ ID NO: 3, and most preferably, a gene represented by SEQ ID NO: 3, but 80% of the nucleotide sequence of SEQ ID NO: 3 It may include a base sequence having sequence homology above, more preferably 90% or more, more preferably 95% or more. When the recombinant protein is expressed, a part of the sequence of the BiP gene may be cut off, leaving only some amino acids.
  • the "cloning site” is a generic term inserted for the purpose of linking/dividing each gene in the vector.
  • the "vesicle signal peptide (ER signal sequence)" is a plant endoplasmic reticulum signal peptide known to those skilled in the art, its kind and amino acid sequence are not limited, and for example, reference may be made to documents such as US 2013/0295065 and WO 2009/158716. have.
  • the "vesicle signal peptide” may preferably be HDEL (His-Asp-Glu-Leu, a polypeptide represented by SEQ ID NO: 7), and may be encoded by a nucleotide sequence represented by SEQ ID NO: 6. .
  • the variant of SEQ ID NO: 6 is included in the scope of the present invention.
  • the gene may include a nucleotide sequence having a sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more with the nucleotide sequence of SEQ ID NO: 6.
  • the binding site of the endoplasmic reticulum signal peptide is characterized in that it is added (or linked) to the C-terminus of a protein for expression or synthesis in plant cells.
  • genes for tags that can be used in addition to the polyhistidine-tag are, for example, Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, His tag, Myc tag, S tag, SBP tag, IgG-Fc Tag, CTB tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, Xpress tag, etc. may be included, and the IgG-Fc tag is derived from human, rat, rabbit or pig. I can.
  • selection marker genes include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and clo Antibiotic resistance genes such as chloramphenicol, aadA genes, etc.
  • the promoters include, for example, pEMU promoter, MAS promoter, histone promoter, Clp promoter, 35S promoter derived from cauliflower mosaic virus, 19S RNA promoter derived from cauliflower mosaic virus, plant actin protein promoter, ubiquitin protein promoter, CMV (Cytomegalovirus) promoter, SV40 (Simian virus 40) promoter, RSV (Respiratory syncytial virus) promoter, EF-1 ⁇ ( Elongation factor-1 alpha) promoter, pEMU promoter, MAS promoter, histone promoter, Clp promoter, etc.
  • the terminator is, for example, nopaline synthase (NOS), rice amylase RAmy1 A terminator, paseolin terminator, The terminator of the octopine gene of Agrobacterium tumafaciens, the rrnB1/B2 terminator of E. coli, etc. may be included, but those listed above are only examples and are not limited thereto.
  • NOS nopaline synthase
  • rice amylase RAmy1 A terminator rice amylase RAmy1 A terminator
  • paseolin terminator paseolin terminator
  • the terminator of the octopine gene of Agrobacterium tumafaciens, the rrnB1/B2 terminator of E. coli, etc. may be included, but those listed above are only examples and are not limited thereto.
  • the recombinant vector is a promoter gene, a polynucleotide encoding a new chaperone binding protein (BIP) signal peptide, a polynucleotide encoding a P32 protein, and a polyhistidine encoding.
  • BIP new chaperone binding protein
  • Polynucleotides and polynucleotides encoding HDEL can be linked in sequence.
  • the recombinant vector according to the present invention includes the nucleotide sequence of SEQ ID NO: 8, and most preferably, the sequence It consists of the nucleotide sequence represented by number 8, but may include a nucleotide sequence having sequence homology of 80% or more, more preferably 90% or more, and more preferably 95% or more with the nucleotide sequence of SEQ ID NO: 8.
  • the present invention provides a transformant transformed with the above recombinant vector.
  • the transformant is preferably microorganisms such as E. coli, Bacillus, Salmonella, yeast, etc., insect cells, animal cells including humans, mice, rats, dogs, monkeys, pigs, horses, cattle Food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum, which may be animals such as animals, Agrobacterium tumorfaciens, plants, etc.; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, and rapeseed; And fruit trees including apple trees, pears, jujubes, peaches, grapes, tangerines, persimmons, plums, apricots, and bananas; And flowers including roses, carn
  • transgenic organism refers to the change of the genetic properties of an organism by the injected DNA
  • “transgenic organism” is a molecular genetic method by injecting an external gene.
  • the prepared living organism preferably it is a living organism transformed by the recombinant expression vector of the present invention, and the living organism is not limited as long as it is a living organism such as microorganisms, eukaryotic cells, insects, animals, plants, preferably E. coli, Salmonella, Bacillus, yeast, animal cells, mice, rats, dogs, monkeys, pigs, horses, cows, acrobacterium tumourfaciens, plants, etc., but are not limited thereto.
  • the term "plant” may be used without limitation as long as it is a plant capable of mass-producing a protein containing an antigen of the present invention, but more specifically, tobacco, Arabidopsis, corn, rice, soybean, canola, alfalfa, sunflower , Alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, lettuce and pepper may be selected from the group consisting of, preferably tobacco.
  • Tobacco in the present invention is not particularly limited as long as it is a plant of the genus Tobacco (Nicotiana genus) and is capable of overexpressing a protein, and the present invention is carried out by selecting an appropriate variety according to the purpose of the transformation method and mass production of the protein.
  • I can.
  • Nicotiana benthamiana L. or Nicotiana tabacum cv. Varieties such as xanthi can be used.
  • the transformants are transformation, transfection, Agrobacterium-mediated transformation method, particle gun bombardment, sonication, electroporation. ) And PEG (polyethylen glycol)-mediated transformation method, etc., but there is no limitation as long as it is a method capable of injecting the vector of the present invention.
  • the present invention provides a P32 recombinant protein (antigen) of African swine fever virus produced using the recombinant vector according to the present invention.
  • the P32 recombinant protein may be water-soluble. More specifically, the recombinant P32 protein expressed in a plant may be 95%, 96%, 97%, 98%, 99% or 100% dissolved in a water-soluble fraction.
  • the P32 recombinant protein may be isolated and purified with a purity of 90% or more. More specifically, the recombinant P32 protein expressed in plants is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, when using a conventional separation and purification method. Recombinant P32 protein of 99% or 100% purity can be obtained.
  • the present invention provides a composition or diagnostic kit for diagnosing African swine fever, comprising as an active ingredient the P32 recombinant protein (antigen) of African swine fever virus produced using the recombinant vector according to the present invention. do.
  • the term “diagnosis” refers to confirming the existence or possibility of invention of a pathological condition.
  • the present invention is particularly useful for diagnosis of African swine fever.
  • the P32 antigen produced by the recombinant vector of the present invention it is possible to detect a specific antibody produced in the body of an individual infected with African swine fever. That is, the P32 antigen can be used as a useful indicator (diagnostic marker) for diagnosing African swine fever.
  • the term “antigen” refers to all substances that cause an immune response in the body, and preferably, viruses, compounds, bacteria, pollen, cancer cells, etc. or some peptides or proteins thereof, or immunity in the body Any material that can cause a reaction is not limited thereto.
  • the present invention uses the recombinant African swine fever virus P32 protein produced using the recombinant vector according to the present invention as an antigen to conduct antigen-antibody reactions in biological samples derived from animals other than humans. It provides a method for diagnosing African swine fever, comprising the step of detecting an antibody against the African swine fever virus.
  • the present invention comprises the steps of (a) transforming the recombinant vector according to the present invention into a plant; And (b) separating and purifying the recombinant antigen from the plant. It provides a method for producing a recombinant antigen for diagnosis of African swine fever.
  • the present invention comprises the steps of (a) transforming the recombinant vector according to the present invention into a plant; (b) separating and purifying the recombinant antigen from the plant; And (c) preparing a diagnostic composition or a diagnostic kit using the isolated/purified recombinant antigen, providing a method for preparing a composition or kit for diagnosing African swine fever.
  • Example 1 Preparation of a recombinant vector expressing the P32 antigen of African swine fever virus
  • a recombinant plant expression vector was constructed so that the plant could express the African swine fever virus antigen P32 protein (known to be involved in the internalization of the virus in the early stages of the infectious cycle).
  • P32 protein known to be involved in the internalization of the virus in the early stages of the infectious cycle.
  • gene information on the P32 protein of the African swine fever virus was obtained, and a gene encoding the P32 protein (SEQ ID NO: 1) was synthesized with a sequence optimized for expression in plants.
  • SEQ ID NO: 3 a biP (chaperone binding protein) signal peptide
  • a polynucleotide encoding a P32 antigen recombinant protein (SEQ ID NO: 1), a polynucleotide encoding a His-tag consisting of 6 consecutive histidines (SEQ ID NO: 4) and HDEL (His-Asp- Glu-Leu) polynucleotide encoding the peptide (SEQ ID NO: 6) was sequentially linked to prepare a plant expression vector of the P32 protein of the African swine fever virus.
  • the plant expression vector prepared in Example 1 was transformed into an Agrobacterium LBA4404 strain using an electrophoration method.
  • the transformed Agrobacteria were cultured with shaking for 16 hours at 28°C in 5 mL of YEP liquid medium (yeast extract 10 g, peptone 10 g, NaCl 5 g, kanamycin 50 mg/L, rifampicin 25 mg/L). Then, 1 mL of the primary culture was inoculated into 50 mL of new YEP medium and cultured with shaking at 28°C for 6 hours.
  • the cultured Agrobacteria were collected by centrifugation (7,000 rpm, 4°C, 5 minutes), and then infiltration buffer [10 mM MES (pH) so that the absorbance (OD) value was 1.0 at a wavelength of 600 nm. 5.7), 10 mM MgCl2, 200 ⁇ M acetosyringon].
  • the Agrobacterial suspension was injected into the back side of Nicotiana benthamiana leaves using a syringe from which the injection needle was removed, and agro-infiltration was performed.
  • the P32 recombinant protein of the African swine fever virus of the present invention was well purified without major alterations or alterations compared to the original protein. These results confirm that when the protein is expressed in plants, a problem in which the sugar structure is changed and production efficiency is not found. It is confirmed that the P32 recombinant protein of the African swine fever virus according to the present invention is well produced in plants. This is the result of confirmation.
  • Example 2.2 Using the P32 antigen protein prepared in Example 2.2, a prototype antibody serum diagnostic kit was prepared, and reactivity and sensitivity tests were conducted with serum provided by the Spanish ASF standard laboratory.
  • the ASF diagnostic kit of the present invention made of the recombinant P32 antigen protein was 100% identical in the negative and positive results as in the sample results provided, and the positive minimum A positive test was confirmed in the serum set to (Fig. 4, limi; Table 1, positive limit control Ref. serum), and it was confirmed that the specificity and sensitivity were excellent.
  • Negative serum 1 (Ne 1) X voice Same 14 Negative serum 2 (Ne 2) X voice Same 15 Negative serum 3 (Ne 3) X voice Same 16 Negative serum 4 (Ne 4) X voice Same 17 Negative serum 5 (Ne 5) X voice Same 18 Negative serum 6 (Ne 6) X voice Same 19 Negative serum 7 (Ne 7) X voice Same 20 Negative serum 8 (Ne 8) X voice Same
  • the recombinant African swine fever virus antigen of the present invention not only enables efficient production using plants, but also has high water solubility, so it is easy to separate and purify, and also, diagnostic sensitivity and specificity ) Is high, so the composition and kit for diagnosing African swine fever prepared using it are expected to be of great industrial value because it can diagnose an individual infected with African swine fever early.

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Abstract

La présente invention concerne un vecteur recombinant portant un polynucléotide codant pour la protéine P32 du virus de la peste porcine africaine, un transformant transformé avec le vecteur recombinant, et une composition et un kit pour le diagnostic de la peste porcine africaine, comprenant chacun une protéine antigénique P32 d'un virus de la peste porcine africaine isolée à partir du transformant.
PCT/KR2020/007762 2019-06-17 2020-06-16 Vecteur recombinant pour produire un antigène pour le diagnostic de la peste porcine africaine et son utilisation WO2020256372A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2020540527A JP7164894B2 (ja) 2019-06-17 2020-06-16 アフリカ豚熱の診断のための抗原生産用組み換えベクター及びその使用
EP20739840.5A EP3835425A4 (fr) 2019-06-17 2020-06-16 Vecteur recombinant pour produire un antigène pour le diagnostic de la peste porcine africaine et son utilisation
US16/964,629 US20230287440A1 (en) 2019-06-17 2020-06-16 Recombinant vector for producing antigen for diagnosis of african swine fever and use therof
CN202080001357.5A CN112424366B (zh) 2019-06-17 2020-06-16 用于生产用于诊断非洲猪瘟的抗原的重组载体及其用途

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CN116286677B (zh) * 2023-04-07 2023-12-12 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 一种非洲猪瘟病毒毒株及其应用

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